A sensitive and specific high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the quantitative determination of gemcitabine (dFdC) and its metabolite 2',2'-difluorodeoxyuridine (dFdU) is presented. A 200-microL aliquot of human plasma was spiked with a mixture of internal standards, didanosine, lamivudine and fludarabine, and extracted using solid-phase extraction. Dried extracts were reconstituted in 1 mM ammonium acetate/acetonitrile (97:3, v/v) and 10-microL volumes were injected onto the HPLC system. Separation was achieved on a 150 x 2.1 mm C18 bonded phase endcapped with polar groups (Synergi Hydro-RP column) using an eluent composed of 1 mM ammonium acetate (pH 6.8)/acetonitrile (94:6, v/v). Detection was performed by positive ion electrospray ionization followed by MS/MS. The assay quantifies a range from 0.5 to 1000 ng/mL for gemcitabine and from 5 to 10,000 ng/mL for dFdU using 200 microL of human plasma sample. Validation results demonstrate that gemcitabine and dFdU concentrations can be accurately and precisely quantified in human plasma. This assay is used to support clinical pharmacologic studies with gemcitabine.
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